We have continued to explore the role that reactive oxygen species (ROS)play in signal transduction pathways. We have previously shown that a variety of growth factors and cytokines induce the generation of ROS following ligand binding. Our studies suggest that the pathway leading to ROS generation involves the activation of the small GTP-binding proteins rac1. This has led us to explore the role of this family of proteins in redox regulation. We have also explored the role that ROS play in apoptosis, and more recently in replicative senescence and aging. In the last year we have been able to demonstrate that oxidants function as specific regulators of pathways linked to aging. In particular, we have demonstrated a relationship between forkhead proteins, a gene family that regulates longevity in C. elegans, p66shc the only gene known to regulate longevity in mammalian cells, and intracellular ROS, thought to regulate aging across a diverse number of species. We have also derived a genomic screening strategy to isolate gene products that regulate mitochondrial oxidant production and potentially regulate cellular and organismal life span. This has led to candidate gene identification of proteins that can positively or negatively regulate lifespan. We have also begun to explore the field of energy signal tranduction, and whether intracellular ROS function as messengers between the mitochondria and the rest of the cell. In addition, this year we have pursued an in vitro to identify direct targets of oxidant stress. We have developed a new assay to allow for the rapid isolation and subsequent identification by mass spectroscopy of proteins in which glutathione has been added in a covalent fashion. We are attempting to understand the physiological relevance of glutathiolation in various signaling pathways. Using this system we have demonstrated that both apoptotic and growth factor stimulation leads to accumulation of a discrete set of proteins in which the reactive cysteine has been covalently modified by glutathione addition. The further identification of these proteins should provide important information on the specificity of oxidant signalling.